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WO1994023744A1 - Vaccin contenant un cytomegalovirus de recombinaison - Google Patents

Vaccin contenant un cytomegalovirus de recombinaison Download PDF

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WO1994023744A1
WO1994023744A1 PCT/US1994/004180 US9404180W WO9423744A1 WO 1994023744 A1 WO1994023744 A1 WO 1994023744A1 US 9404180 W US9404180 W US 9404180W WO 9423744 A1 WO9423744 A1 WO 9423744A1
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amino acid
fragment
ser
val
leu
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PCT/US1994/004180
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English (en)
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Stanley A. Plotkin
Robert P. Ricciardi
Eva Gonczol
Klara Berencsi
Robert F. Rando
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The Wistar Institute Of Anatomy And Biology
THE GOVERNMENT OF THE UNITED STATES OF AMERICA on behalf of THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
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Priority to EP94915386A priority Critical patent/EP0708658A4/fr
Publication of WO1994023744A1 publication Critical patent/WO1994023744A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention refers generally to a recombinant human cytomegalovirus vaccine, and more specifically to a subunit vaccine containing fragments of a HCMV major glycoprotein complex subunit gB gene.
  • Cytomegalovirus is one of a group of highly host specific herpes viruses that produce unique large cells bearing intranuclear inclusions.
  • the envelope of the human cytomegalovirus (HCMV) is characterized by a major glycoprotein complex recently termed gB or gCI, which was previously referred to as gA.
  • HCMV causes cytomegalic inclusion disease and has been associated with a syndrome resembling infectious mononucleosis in adults. It also induces complications in immunocompromised individuals.
  • CMV infection in utero is an important cause of central nervous system damage in newborns. Although the virus is widely distributed in the population, about 40% of women enter pregnancy without antibodies and thus are susceptible to infection. About 1% of these women undergo primary infection in utero .
  • Classical cytomegalic inclusion disease is rare; however, a proportion of the infected infants, including those who were symptom-free, are subsequently found to be mentally retarded.
  • Preliminary estimates based on surveys of approximately 4,000 newborns from several geographical areas indicate that the virus causes significant damage of the central nervous system leading to mental deficiency in at least 10%, and perhaps as high as 25%, of infected infants.
  • HCMV in humans has also been observed to cause serious complications and infections in the course of organ transplantations, especially with kidney and liver transplants.
  • HCMV vaccines have been developed or are in the process of development. Vaccines based on live attenuated strains of HCMV have been described. [See, e.g., S. A. Plotkin et al, Lancet, 1:528-30 (1984); S. A. Plotkin et al, J. Infect. Pis.. 134:470-75 (1976); S. A. Plotkin et al, "Prevention of Cytomegalovirus Pisease by Towne Strain Live Attenuated Vaccine", in birth Pefects, Original Article Series, 20(1) :271-287 (1984); J. P. Glazer et al, Ann. Intern. Med..
  • a proposed HCMV vaccine using a recombinant vaccinia virus expressing HCMV glycoprotein B has also been described. [See, e.g., Cranage, M. P. et al, EMBO J.. 5:3057-3063 (1986).] However, vaccinia models for vaccine delivery are believed to cause local reactions. Additionally, vaccinia vaccines are considered possible causes of encephalitis.
  • Adenoviruses have been developed previously as efficient heterologous gene expression vectors. For example, an adenovirus vector has been employed to express herpes simplex virus glycoprotein gB [P. C. Johnson et al, Virol..
  • Adenoviruses have also been found to be non-toxic as vaccine components in humans [See, e.g., E. T. Takajuji et al, J.
  • the present invention provides a non-defective recombinant adenovirus containing a fragment of a gB subunit of the HCMV free from association with any additional human proteinaceous material.
  • the HCMV subunit is under the control of regulatory sequences capable of expressing the HCMV gB subunit fragment in vitro and in vivo.
  • Another aspect of the present invention is a vaccine composition comprising a non-defective recombinant adenovirus, as described above.
  • the invention provides a method of vaccinating a human against HCMV comprising administering to the patient the recombinant adenovirus containing the subunit gene encoding a gB protein fragment in a vaccine composition.
  • This method of presenting these HCMV gene fragments to a vaccinate is particularly capable of eliciting an immune response.
  • the invention provides an adenovirus-produced gB subunit fragment, which fragment may also form vaccine compositions to protect humans against HCMV.
  • the preferred fragment comprises about amino acids 1 to about 303 of the gB protein SEQ IP NO:2, gB-.. 3(a .
  • Fig. IA illustrates diagrammatically the cloning of the gB gene into the early region 3 (E3) transcription unit of Ad5. Represented are the 3.1kb fragment containing the gB gene by the open box; the adenovirus sequences extending from 59.5 to 100 mu (except for the deletion of the 78.5 to 84.7 mu length) by the filled portion of the circle: the large BamHI fragment of the pBR322 by the thin line of the circle.
  • restriction enzymes are identified as follows: X is Xbal, B is BamHI.
  • Fig. IB illustrates diagrammatically the construction of the recombinant adenovirus virus Ad5/gB, containing the gB gene of the Towne strain of HCMV described in Example 1.
  • This figure shows the 59.5 mu to 76 mu region where homologous recombination occurs (as indicated by the crossed lines) between wild type Ad5 viral sequence and the adenovirus sequences present on the pAd5 plasmid containing the gB gene.
  • the plaque purified recombinant virus retains the cloning Xbal sites and'the direction of transcription of the gB gene from the E3 promoter is indicated by the bent arrow. Restriction enzymes are as identified above.
  • the present invention provides novel immunogenic components for HCMV which comprise an adenovirus expression system capable of expressing a selected HCMV subunit gene fragment in vivo.
  • the selected subunit fragment for use in an immunogenic composition may be expressed in, and isolated from, the recombinant adenovirus expression system.
  • any adenovirus strain capable of replicating in mammalian cells in vitro may be used to construct an expression vector for the selected HCMV subunit.
  • a preferred expression system involves a non-defective adenovirus strain, including, but not limited to, adenovirus type 5.
  • other desirable adenovirus strains may be employed which are capable of being orally administered, for use in expressing the CMV subunit in vivo.
  • Such strains useful for in vivo production of the subunit in addition to adenovirus-5 strains include adenovirus type 4, 7, and 21 strains. [See, e.g., Takajuji et al, cited above]. Appropriate strains of adenovirus, including those identified above and those employed in the examples below are publicly available from sources such as the American Type Culture Collection, Rockville, Maryland.
  • a number of strains of isolated human CMV may be employed from which a desired gB subunit is derived.
  • the Towne strain of CMV a preferred strain for use in preparation of a vaccine of this invention because of its broad antigenic spectrum and its attenuation, was isolated from the urine of a two month old male infant with cytomegalic inclusion disease (symptoms - central nervous system damage and hepatosplenomegaly) .
  • This strain of CMV was isolated by Stanley A. Plotkin, M.P. and is described in J. Virol.. 11 (6) : 991 (1973) .
  • This strain is freely available from The istar Institute or from the ATCC under accession number VR-977.
  • other strains of CMV useful in the practice of this invention may be obtained from depositories like the ATCC or from other institutes or universities.
  • the HCMV subunit may be produced in vitro by recombinant techniques in large quantities sufficient for use in an immunogenic composition or subunit vaccine.
  • the recombinant adenovirus containing the subunit may itself be employed as an immunogenic or vaccine component, capable of expressing the subunit in vivo .
  • the presently preferred subunit proteins for use in the present invention are the HCMV gB subunit fragments.
  • One embodiment of the present invention provides a replication competent (non-defective) adenovirus vector carrying a fragment of the HCMV gB gene which contains a CTL epitope and/or B cell epitope.
  • a preferred gene fragment encodes about amino acid 1 to about amino acid 303 of the gB subunit protein SEQ IP NO:2.
  • Another suitable fragment of gB SEQ IP NO:2 is the fragment spanning about amino acid 1 to about amino acid 700 of SEQ IP NO:2.
  • Still another suitable gB fragment spans about amino acid 1 to about amino acid 465 of SEQ IP NO:2.
  • HCMV subunit may be employed in a vaccine according to the teachings of the present invention.
  • sequences of the subunits of two HCMV strains have been published [See, e.g., Mach et al, J. Gen. Virol.. 67_ ⁇ 1461-1467 (1986); Cranage et al, (1986) cited above; and Spaete et al, Virol.. 167:207-225 (1987) .
  • These subunit sequences can therefore be chemically synthesized by conventional methods known to one of skill in the art, or the sequences purchased from commercial sources.
  • the recombinant adenovirus of the present invention may also contain multiple copies of the HCMV subunit.
  • the recombinant virus may contain more than one HCMV subunit type, so that the virus may express two or more HCMV subunits, subunit fragments, or immediate early antigens and subunits together.
  • the CMV subunit sequence is preferably inserted in an adenovirus strain under the control of an expression control sequence in the virus itself.
  • the adenovirus vector of the present invention preferably contains other sequences of interest in addition to the HCMV subunit. Such sequences may include regulatory sequences, enhancers, suitable promoters, secretory signal sequences and the like.
  • sequences may include regulatory sequences, enhancers, suitable promoters, secretory signal sequences and the like.
  • the techniques employed to insert the subunit sequence into the adenovirus vector and make other alterations in the viral PNA, e.g., to insert linker sequences and the like, are known to one of skill in the art. See, e.g., T. Maniatis et al, "Molecular Cloning.
  • adenovirus expression vectors for expression of an HCMV subunit protein is within the skill of the art.
  • Example 3 below provides construction details for the non-defective adenovirus containing these gB fragments.
  • the recombinant adenovirus itself may be used directly as an immunogen or a vaccine component.
  • the recombinant adenovirus containing the HCMV subunit, e.g., the gB subunit fragment, is introduced directly into the patient by vaccination.
  • the recombinant virus when introduced into a patient directly, infects the patient's cells and produces the CMV subunit in the patient's cells.
  • the inventors have found that this method of presenting these HCMV genes to a vaccinate is particularly capable of eliciting an immune response.
  • Examples 5 and 6 demonstrate the ability of a recombinant adenovirus containing the gB fragment, amino acid 1-303 of SEQ IP NO:2, to induce a gB-specific, protective CTL response in mice.
  • adenovirus recombinants as immunogens capable of inducing a CTL response is surprising in view of the results obtained in the same assays of the examples with other known virus types, which have been used in vaccines previously.
  • the recombinant viral vector containing the CMV subunit protein e.g., the gB ⁇ r ⁇ subunit fragment
  • it may be infected into a suitable host cell for in vitro expression.
  • the infection of the recombinant viral vector is performed in a conventional manner.
  • Suitable host cells include, without limitation, mammalian cells and cell lines, e.g., A549 (human lung carcinoma) or 293 (transformed human embryonic kidney) cells.
  • the host cell once infected with the recombinant virus of the present invention, is then cultured in a suitable medium, such as Minimal Essential Medium (MEM) for mammalian cells.
  • MEM Minimal Essential Medium
  • the culture conditions are conventional for the host cell and allow the subunit, e.g., gB W( ⁇ subunit fragment, to be produced either intracellularly, or secreted extracellularly into the medium.
  • Conventional protein isolation techniques are employed to isolate the expressed subunit from the selected host cell or medium.
  • the subunit When expressed in vitro and isolated from culture, the subunit, e.g., gB ⁇ , may then be formulated into an appropriate vaccine composition.
  • Such compositions may generally contain one or more of the recombinant CMV subunits.
  • the present invention also includes a method of vaccinating humans against human CMV infection with the recombinant adenovirus vaccine composition.
  • This vaccine composition is preferably orally administered, because adenoviruses are known to replicate in cells of the stomach. Previous studies with adenoviruses have shown them to be safe when administered orally [see, e.g., Collis et al, cited above].
  • the present invention is not limited by the route of administration selected for the vaccine.
  • a dosage of between 10 5 and 10 8 plaque forming units may be used. Additional doses of the vaccines of this invention may also be administered where considered desirable by the physician.
  • the dosage regimen involved in the method for vaccination against CMV infection with the recombinant virus of the present invention can be determined considering various clinical and environmental factors known to affect vaccine administration.
  • the vaccine composition may comprise one or more recombinantly-produced human CMV subunit proteins, preferably a fragment of a gB subunit.
  • the in vitro produced subunit proteins may be introduced into the patient in a vaccine composition as described above, preferably employing the oral, nasal or subcutaneous routes of administration.
  • Such an immune response is capable of providing protection against exposure to the whole human CMV microorganism.
  • the dosage for all routes of administration of the in vitro vaccine containing one or more of the CMV subunit proteins is generally greater than 20 micrograms of protein per kg of patient body weight, and preferably between 40 and 80 micrograms of protein per kilogram.
  • adenovirus The utility of the recombinant adenoviruses of the present invention is demonstrated through the use of a novel mouse experimental model which characterizes cytotoxic T lymphocyte (CTL) responses to individual proteins of strictly human-restricted viruses.
  • CTL cytotoxic T lymphocyte
  • the model as used herein is based on the use of two types of recombinant viruses, an adenovirus and a canarypox virus, both expressing a gene of the same HCMV protein.
  • mice are immunized with one recombinant of the invention, and CTL activity is tested in target cells infected with the other recombinant.
  • Examples 4-6 below provide a murine model of the cytotoxic T lymphocyte (CTL) response to the amino acid 1-303 fragment of the glycoprotein B (gB) gene [SEQ IP NO:2] of human cytomegalovirus (HCMV) based on the use of gB-expressing adenovirus (Ad-gB) and several poxvirus recombinants.
  • CTL cytotoxic T lymphocyte
  • gB glycoprotein B
  • Ad-gB gB-expressing adenovirus
  • MHC major histocompatibility complex
  • the gB gene was cloned from the Towne strain of HCMV [Wistar Institute] as follows. The gB gene was first mapped to the 20.5 kb Hind III P fragment of HCMV using oligonucleotides that corresponded to the 5' and 3' termini of the published AP-169 gB sequence [See, Cranage et al (1986), cited above]. The Hind III fragment was cut with Xbal to generate a 9.8 kb fragment. This fragment was then cut with X alll to generate a 3.1 kb fragment. The 3.1 kb Xmalll fragment which contained the gB gene, had Xbal linkers attached to its 5' and 3' termini.
  • pAd5 Bam-B An adenovirus type 5 plasmid, pAd5 Bam-B, which contains the 59.5 - 100 mu region of the Ad5 adenovirus genome cloned into the BamHI site of pBR322 [See, R. L. Berkner et al, Nucl. Acids Res.. 1 ⁇ :6003-6020 (1983) and M. E. Morin et al, cited above] was digested with Xbal to remove the 78.5 mu - 84.7 mu sequences of the Ad5 genome. The 78.5 to 84.7 mu deletion removes most of the coding region of the E3 transcription unit of Ad5 but leaves the E3 promoter intact.
  • Fig. IA provides a diagrammatic illustration of the above description.
  • the 0-76 mu fragment of wild type Ad5 virus was isolated by digesting the viral PNA with EcoRI [See, U. Petterson et al, J. Mol. Biol. , 72:125-130 (1973)]. This fragment was co- transfected with the 59.5 to 100 mu BamHI fragment of pAd5 Bam-B containing the gB gene as described above into human embryonic kidney 293 cells, available from the American Type Culture Collection.
  • the Ad-gB recombinant was generated by overlap recombination between the viral sequences as illustrated in Fig. IB.
  • the gB recombinant virus was plaque purified on human lung carcinoma A549 cells [ATCC CCL185] using standard procedures. Viruses containing both orientations of the gB gene, as determined by Southern blotting, were isolated.
  • the recombinant containing the gB gene in the same 5• to 3' direction as the adenovirus E3 promoter of the adenovirus type 5 strain is under the transcriptional control of the E3 promoter.
  • the plaque purified recombinant virus retains the cloning Xbal sites.
  • the above-described cloned gB gene is devoid of its natural promoter according to the PNA sequence of gB identified in Spaete et al, (1987) , cited above.
  • Example 2 Production of the Full-Length gB Subunit
  • the adenovirus gB plasmid construct and the Ad5 mu 0-76 PNA of Example 1 were cotransfected into 293 cells, human cells transformed by adenovirus 5 early genes [See, Graham et al, J. Gen. Virol. ,36:59-72 (1977); and ATCC CRL1573] employing conventional procedures.
  • This transfection generated a functional recombinant virus by homologous overlap recombination as shown in
  • Southern blot analysis confirmed the presence of an adenovirus, type 5, containing the HCMV gB subunit (referred to as either Ad-5/gB or Ad-gB) recombinant virus which was subsequently purified by plaque purification using standard procedures.
  • the recombinant virus AP-5/gB expresses gB subunit protein as determined by conventional assays, i.e., immunofluorescence on fixed cells and by Western blot using monospecific guinea pig antiserum and monoclonal antibodies to gB protein [See, e.g., T. Maniatis et al, cited above].
  • the Ad-5/gB recombinant also referred to as Ad-gB, is also described in applicant's publication [Marshall et al., J. Infect. Pis.. 162:1177-1181 (1990)] published after the filing date of the original parent application from which this application claims priority.
  • Ad-gBi. 303 and Ad-gB M55 recombinant viruses were constructed by overlap recombination as described for Ad- gB in Example 2 above. Briefly, in order to clone the subfragments of the gB gene, five oligonucleotide primers for polymerase chain reactions (PCR) were synthesized.
  • PCR polymerase chain reactions
  • the primers were designed to anneal with various portions of the gB PNA sequence and promote amplification of the gene.
  • all of the oligonucleotide primers were engineered to contain an Xba I site so that the PCR product could be digested with this enzyme in order to facilitate cloning into the pAd-5 vector.
  • 5' gB primer SEQ IP NO:3:
  • oligonucleotides correspond to the following nucleotide positions of the HCMV gB gene (Towne strain) as reported by Spaete et al, Virology. 167:207-225 (1988).
  • SEQ IP N0:3 corresponds to nucleotide positions 895 to 922 in the sense orientation; SEQ IP NO:4 to nucleotide positions 3090 to 3067 anti-sense; SEQ IP NO:5 to nucleotide positions 2375 to 2350 anti-sense; SEQ IP NO:6 to nucleotide positions 1877 to 1847 anti-sense; and SEQ IP NO:7 to nucleotide positions 1432 to 1400 anti- sense.
  • the specific segments or fragments of the gB gene were amplified using the Perkin-Elmer AmplitaqTM kit by mixing 400 ng of the 5' gB primer with each of the 3' primers separately (400 ng of each) and 0.1 ⁇ g of purified HCMV genomic PNA or 0.1 ⁇ g of previously cloned intact gB gene (see Example 2) .
  • the final reaction mixture was 100 / xL and the thermocycling conditions were 94°C, 1 minute; 52°C, 1 minute; 72°C, 1 minute, repeated for a total of 35 cycles.
  • Amplified PNA was purified by cutting the proper PNA fragment out of a 1.2% agarose gel, digested with Xbal.
  • Example 4 CTL Assays A. Recombinant Viruses Used
  • Wild-type human adenovirus type 5 WT-Ad
  • Ad-gB Ad-gB
  • E3-deleted adenovirus type 5 mutant lacking the Xbal P fragment of adenovirus PNA was constructed by overlap recombination, using plasmid pAd-5 mu 59.5-100, which was deleted in E3 sequences (mu 78.5-84) using the techniques described in Example 1, and pAd-5 mu 0-75.9 [G. S. Marshall et al, J. Infect. Pis.. 162:1177-1181 (1990), hereby incorporated by reference].
  • the vaccinia WR strain [obtained from Pr. Enzo Paoletti, Virogenetics Corp, Troy, NY] was used to develop a recombinant expressing HCMV-gB ( (VacW)-gB) .
  • This recombinant was derived using a strategy similar to that described for the VacC-gB recombinant (Gonczol et al. , cited above) .
  • a canarypox recombinant [ALVAC-CMV (VCP139) which is subsequently referred to as Cp-gB] expressing the HCMV-gB gene was constructed using a strategy similar to that described for a canarypox-rabies recombinant in Taylor et al., Vaccine. 9.:190-193 (1991) [also obtained from Pr. Enzo Paoletti] .
  • HCMV Downe strain glycoprotein B glycoprotein B
  • canarypox donor plasmid consisting of a polylinker flanked by genomic sequence from which a nonessential gene was specifically deleted (at a unique EcoRI site within a 3.3 kbp PvuII subgenomic fragment of canarypox PNA) .
  • Expression of the gB protein gene was placed under the transcriptional control of an early/late vaccinia virus promoter (H6) previously described [Percus et al., J. Virol.. .62:3829-3835 (1989)].
  • H6 early/late vaccinia virus promoter
  • Cp-gB recombinant and parental canarypox virus were propagated on primary chick embryo fibroblasts.
  • B. Expression of the gB protein in Cp-gB recombinant virus Chicken embryo fibroblast (CEF) cells [ATCC CRL 1590] infected with either Cp-gB or with the parental wild-type canarypox (WT-Cp) virus preparations were analyzed by Western blot assay using the 4A guinea-pig serum directed against the gB protein.
  • a diffuse band at the 140 kPa position and a double band of 55 and 58 kPa were detected in both Cp-gB-infected CEF cells and in HCMV-infected MRC-5 cells.
  • the presence of these gB-specific proteins presumably representing the glycosylated 140 kPa precursor and the differentially glycosylated cleavage products (55 and 58 kPa) indicates that the Cp-gB recombinant expresses the inserted gB gene.
  • the slight difference between the mobility of 55 and 58 kPa cleavage products of control and recombinant gB may reflect different glycosylation patterns.
  • Ad-gB and WT-Ad were purified by CsCl gradient centrifugation.
  • VacC-gB, VacW-gB and WT-Vac were purified by sucrose gradient centrifugation, and Cp-gB and WT-Cp were concentrated on sucrose cushion.
  • mice Six- to 8-week-old female BALB/c and CBA mice (from Harlan Sprague-Pawley and Jackson) and 12-week-old male BALB/k mice (from The Wistar Institute Animal Facility) were immunized intraperitoneally (i.p.) with the recombinant viruses described above at 1-5 x IO 8 pfu unless otherwise stated.
  • spleens were aseptically removed and cell suspensions were prepared by gently pressing the spleens through a stainless steel mesh.
  • CP4 or CP8 cells For in vitro depletion of CP4 or CP8 cells, 3 x 10 6 spleen cells were incubated with anti-mouse CP4 monoclonal antibody (MAb) [Pharmingen; Cat.3:01061 P; 20 ⁇ g/3xl0 6 cells] or CP8 MAb [Accurate; Cat.#:CL-8921; diluted 1:4] for 60 minutes at 4°C, and further incubated in the presence of rabbit complement [Accurate; Low-tox M; diluted 1:10] for 30 minutes at 37°C. The cells were washed twice and used as effector cells in a 51 Cr-release test.
  • MAb monoclonal antibody
  • mice MC57 (H- 2 b ) cells [also termed MC-57G, P.P. Aden et al, Im unogenetics, 2:209-221 (1976)] and mouse NCTC clone 929 (H-2 k ) cells [ATCC CCL 1] were used as target cells.
  • the HCMV neutralization titer of mouse sera was determined on MRC-5 cells [ATCC CCL 171] by the microneutralization method as described in Gonczol et al., J. Virol. Methods. 14:37-41 (1986).
  • Target cells were washed in the modified RPMI 1640 medium described above and 2 x 10 6 cells were labeled with 100 ⁇ Ci of [ 51 Cr]NaCr04 [Amersham, specific activity 250-500 mCi/mg] for 1 hour.
  • the labeled target cells were washed 3 times in phosphate-buffered saline (PBS) and then mixed with the effector cells at various effector:target ratios in triplicate using 96-well U-bottomed microtiter plates and incubated for 4 hours.
  • PBS phosphate-buffered saline
  • Percentage specific 51 Cr release was calculated as: [ (cpm experimental release - cpm spontaneous release) / (cpm maximal release - cpm spontaneous release)] x 100. Standard deviation of the mean of triplicate cultures was less than 10%, and spontaneous release was always less than 25%.
  • This CTL assay is a system in which two types of viral expression vectors, poxvirus and adenovirus, carrying the same fragment of the HCMV-gB gene, are alternately used for immunization of animal or for infection of target cells to show that HCMV-gB fragment is an inducer of CTL in mice.
  • poxvirus and adenovirus carrying the same fragment of the HCMV-gB gene
  • Ad-gB j . 303 and Ad-gB WJ5 recombinant viruses were constructed as described in Example 3 above.
  • CBA mice were immunized i.p. with IO 8 pfu of the Ad-gB, Two weeks later spleen cells were restimulated in vitro with Ad-gB infected autologous spleen cells and tested for ability to lyse Wt-Ad, Vac-gB or Wt-Vac infected L929 (MHC-class I matched) cells.
  • CBA mice were immunized with 1 x 10 8 pfu of Wt-Ad, Ad5 ⁇ 3 (an E3 deleted mutant virus, the parental strain of the recombinant viruses) , Ad-gB, Ad-gB ⁇ or Ad-gB ⁇ _ 155 .
  • Ad-gB an E3 deleted mutant virus, the parental strain of the recombinant viruses
  • Ad-gB ⁇ an E3 deleted mutant virus, the parental strain of the recombinant viruses
  • Ad-gB Ad-gB ⁇
  • Ad-gB ⁇ _ 155 Ad-gB
  • VacWR-gB a neurovirulent vaccinia strain expressing the HCMV-gB protein
  • Ad-gB and Ad-gB ⁇ -immunized mice survived (92% and 95% survival, respectively) , while all of the Ad-gB j .i jj -i munized mice died, indicating a protection epitope on the N-terminal part of the gB protein between amino acid 155 and 303.
  • HCMV major glycoprotein complexes e.g., gcll or gcIII, or immediate-early antigens
  • gcll or gcIII e.g., immediate-early antigens
  • immediate-early antigens may be expressed in a non- defective adenovirus recombinant in the same manner as described above for subunit gB fragment.
  • GGT ACG GAT CTT ATT CGC TTT GAA CGT AAT ATC GTC TGC ACC 336 Gly Thr Asp Leu lie Arg Phe Glu Arg Asn lie Val Cys Thr 100 105 110
  • TAC ATC CAC ACC ACT TAT CTG CTG GGC AGC AAC ACG GAA TAC 504 Tyr lie His Thr Thr Tyr Leu Leu Gly Ser Asn Thr Glu Tyr 155 160 165
  • AAG ATC TTC ATC GCC GGC AAC TCG GCC TAC GAG TAC GTG GAC 1890 Lys lie Phe lie Ala Gly Asn Ser Ala Tyr Glu Tyr Val Asp 620 625 630
  • GCC ATT GGG GCC GTG GGT GGC GCG GTG GCC TCC GTG GTC GAA 2226 Ala lie Gly Ala Val Gly Gly Ala Val Ala Ser Val Val Glu 730 735 740
  • ATC ATC CTC GTG GCC ATA GCC GTC GTC ATT ATC ATT TAT TTG 2310 lie lie Leu Val Ala lie Ala Val Val lie lie lie Tyr Leu 760 765 770
  • Val Val Asp lie Ser Pro Phe Tyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285
  • Ser lie Ser Thr Val Asp Ser Met lie Ala Leu Asp lie Asp Pro Leu

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Abstract

La présente invention concerne un système d'expression d'un adénovirus non défectif utilisé pour l'expression d'un fragment immunogénique de la sous-unité gb du cytomégalovirus humain (HCMV), ledit adénovirus de recombinaison exprimant le HCMV étant utile en tant que vaccin.
PCT/US1994/004180 1993-04-16 1994-04-15 Vaccin contenant un cytomegalovirus de recombinaison WO1994023744A1 (fr)

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US5591439A (en) * 1989-03-24 1997-01-07 The Wistar Institute Of Anatomy And Biology Recombinant cytomegalovirus vaccine
EP0837928A1 (fr) * 1995-06-06 1998-04-29 Virogenetics Corporation Cytomegalovirus-poxvirus de recombinaison, compositions et utilisations
US6086876A (en) * 1997-02-07 2000-07-11 The Wistar Insitute Methods and compositions for the inhibition of interleukin-12 production
US6448389B1 (en) 1996-04-23 2002-09-10 The Wistar Institute Of Anatomy And Biology Human cytomegalovirus DNA constructs and uses therefor
WO2003000720A1 (fr) * 2001-06-26 2003-01-03 The Council Of The Queensland Institute Of Medical Research Nouveaux epitopes des lymphocytes t cytotoxiques du cytomegalovirus humain (hcmv), polyepitopes, compositions comprenant ces derniers et utilisations diagnostiques, prophylactiques et therapeutiques correspondantes
US6586192B1 (en) 1998-05-29 2003-07-01 Thomas Jefferson University Compositions and methods for use in affecting hematopoietic stem cell populations in mammals
WO2004020405A2 (fr) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Proteines de fusion de transferrine modifiees
WO2004020454A2 (fr) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Proteines de fusion modifiees a transferrine comprenant des domaines aminotransferrine ou carboxy terminal dupliques
US6846486B1 (en) 2000-02-24 2005-01-25 Advanced Biotherapy Concepts, Inc. Method of treating allergy by administering an anti-histamine antibody
US7094569B2 (en) 2001-05-24 2006-08-22 Soogyun Kim Hair follicle growth factor proteins
WO2007059513A2 (fr) 2005-11-15 2007-05-24 The Children's Hospital Of Philadelphia Compositions et procedes de modulation d’hemostase
WO2007068803A1 (fr) 2005-12-14 2007-06-21 Licentia Ltd Nouveau facteur neurotrophique et utilisations de celui-ci
US7314618B2 (en) 2000-07-19 2008-01-01 Advanced Research And Technology Institute Antibodies to fibroblast growth factor (FGF23)
US7371846B2 (en) 1999-05-24 2008-05-13 The Trustees Of The University Of Pennsylvania CD4-independent HIV envelope proteins as vaccines and therapeutics
US7608433B2 (en) 2005-02-09 2009-10-27 Idexx Laboratories Method of detection of classical swine fever
WO2009133247A1 (fr) 2008-04-30 2009-11-05 Licentia Oy Facteur neurotrophique manf et ses utilisations
WO2010060035A1 (fr) 2008-11-21 2010-05-27 The Children's Hospital Of Philadelphia Facteur v de serpent et procédés d'utilisation en tant que procoagulant
US7754482B2 (en) 2004-05-27 2010-07-13 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses therefor
EP2384764A2 (fr) 2006-02-23 2011-11-09 The Children's Hospital of Philadelphia Compositions et méthodes pour moduler une hémostase au moyen de différentes formes de facteur V activé
WO2012074868A2 (fr) 2010-12-03 2012-06-07 Ms Technologies, Llc Expression optimisée de molécules d'acide nucléique codant pour la résistance au glyphosate dans cellules végétales
EP2479189A1 (fr) 2006-12-12 2012-07-25 Biorexis Pharmaceutical Corporation Bibliothèques de protéines hybrides de transferrine
EP2551281A1 (fr) 2007-10-25 2013-01-30 Nevalaita, Lina Variants d'épissage de GDNF et leurs utilisations
WO2013049804A1 (fr) 2011-09-30 2013-04-04 The Children's Hospital Of Philadelphia Compositions et procédés pour moduler l'homéostasie
WO2014191630A2 (fr) 2013-05-28 2014-12-04 Helsingin Yliopisto Modèle animal non humain codant pour un gène manf non fonctionnel
US9345677B2 (en) 2003-04-04 2016-05-24 Incyte Corporation Compositions, methods and kits relating to HER-2 cleavage
US9783817B2 (en) 2013-03-04 2017-10-10 Arkansas State University Methods of expressing and detecting activity of expansin in plant cells
WO2018226887A1 (fr) 2017-06-07 2018-12-13 Spark Therapeutics, Inc. Agents d'activation destinés à la transfection de cellules et/ou la production de vecteur raav améliorées
WO2019152957A1 (fr) 2018-02-02 2019-08-08 Arizona Board Of Regents On Behalf Of Arizona State University Lymphocytes t de type adn-récepteur antigénique chimérique pour immunothérapie
WO2022079083A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acide nucléique pour transcription de va-arn
WO2022079082A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acides nucléiques améliorées pour activation de gènes simultanée
WO2023198685A1 (fr) 2022-04-13 2023-10-19 F. Hoffmann-La Roche Ag Procédé de détermination de génomes d'aav
WO2023227438A1 (fr) 2022-05-23 2023-11-30 F. Hoffmann-La Roche Ag Procédé raman de différenciation d'un sérotype de particules aav et d'un état de chargement de particules aav
WO2023232922A1 (fr) 2022-06-03 2023-12-07 F. Hoffmann-La Roche Ag Procédé de production de particules d'aav recombinées
WO2024013239A1 (fr) 2022-07-14 2024-01-18 F. Hoffmann-La Roche Ag Procédé de production de particules de virus adéno-associé recombinant
WO2024056561A1 (fr) 2022-09-12 2024-03-21 F. Hoffmann-La Roche Ag Procédé de séparation de particules de vaa pleines et vides
WO2024165456A1 (fr) 2023-02-07 2024-08-15 F. Hoffmann-La Roche Ag Procédé de détection d'anticorps anti-particules d'aav
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4920209A (en) * 1984-11-01 1990-04-24 American Home Products Corporation Oral vaccines

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5124440A (en) * 1986-11-24 1992-06-23 The Childrens Hospital, Inc. Antibody and T cell recognition sites on glycoproteins comprising the GCI complex of human cytomegalovirus
EP0429470A1 (fr) * 1988-07-01 1991-06-05 The Children's Hospital, Incorporated Reconnaissance de fragments provenant de cytomegalovirus a l'aide d'anticorps
AU4664689A (en) * 1988-12-12 1990-07-10 Children's Hospital Incorporated, The Proteolytic fragments obtained from human cytomegalovirus glycoprotein complex i
DE69027695T2 (de) * 1989-03-24 1997-02-20 Wistar Inst Rekombinanter Cytomegalovirus-Impfstoff

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4920209A (en) * 1984-11-01 1990-04-24 American Home Products Corporation Oral vaccines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP0708658A4 *
THE EMBO JOURNAL, Volume 5, No. 11, issued November 1986, CRANAGE et al.: "Identification of the Human Cytomegalovirus Glycoprotein B Gene and Induction of Neutralizing Antibodies via Its Expression in Recombinant Vaccinia Virus", pages 3057-3063, see entire document. *

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Publication number Priority date Publication date Assignee Title
US5591439A (en) * 1989-03-24 1997-01-07 The Wistar Institute Of Anatomy And Biology Recombinant cytomegalovirus vaccine
EP0837928A4 (fr) * 1995-06-06 2003-01-22 Virogenetics Corp Cytomegalovirus-poxvirus de recombinaison, compositions et utilisations
EP0837928A1 (fr) * 1995-06-06 1998-04-29 Virogenetics Corporation Cytomegalovirus-poxvirus de recombinaison, compositions et utilisations
US6448389B1 (en) 1996-04-23 2002-09-10 The Wistar Institute Of Anatomy And Biology Human cytomegalovirus DNA constructs and uses therefor
US6086876A (en) * 1997-02-07 2000-07-11 The Wistar Insitute Methods and compositions for the inhibition of interleukin-12 production
US6586192B1 (en) 1998-05-29 2003-07-01 Thomas Jefferson University Compositions and methods for use in affecting hematopoietic stem cell populations in mammals
US7371846B2 (en) 1999-05-24 2008-05-13 The Trustees Of The University Of Pennsylvania CD4-independent HIV envelope proteins as vaccines and therapeutics
US6846486B1 (en) 2000-02-24 2005-01-25 Advanced Biotherapy Concepts, Inc. Method of treating allergy by administering an anti-histamine antibody
US7314618B2 (en) 2000-07-19 2008-01-01 Advanced Research And Technology Institute Antibodies to fibroblast growth factor (FGF23)
EP2184296A1 (fr) 2000-07-19 2010-05-12 Advanced Research And Technology Institute, Inc. Facteur de croissance fibroblastique (fgf23) et ses méthodes d'utilisation
US8586317B2 (en) 2000-07-19 2013-11-19 Advanced Research & Technology Institute Methods of diagnosing hypophosphatemic disorders
US7335641B2 (en) 2001-05-24 2008-02-26 Soogyun Kim Method for stimulating hair follicle cell proliferation
US7094569B2 (en) 2001-05-24 2006-08-22 Soogyun Kim Hair follicle growth factor proteins
WO2003000720A1 (fr) * 2001-06-26 2003-01-03 The Council Of The Queensland Institute Of Medical Research Nouveaux epitopes des lymphocytes t cytotoxiques du cytomegalovirus humain (hcmv), polyepitopes, compositions comprenant ces derniers et utilisations diagnostiques, prophylactiques et therapeutiques correspondantes
US7524503B2 (en) 2001-06-26 2009-04-28 The Council Of The Queensland Institute Of Medical Research Human cytomegalovirus (HCMV) cytotoxic T cell epitopes, polyepitopes compositions comprising same and diagnostic and prophylactic and therapeutic uses therefor
WO2004020454A2 (fr) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Proteines de fusion modifiees a transferrine comprenant des domaines aminotransferrine ou carboxy terminal dupliques
WO2004020405A2 (fr) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Proteines de fusion de transferrine modifiees
US9345677B2 (en) 2003-04-04 2016-05-24 Incyte Corporation Compositions, methods and kits relating to HER-2 cleavage
US10286066B2 (en) 2004-05-27 2019-05-14 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses thereof
EP3363907A1 (fr) 2004-05-27 2018-08-22 The Trustees of the University of Pennsylvania Nouvel antigène artificiel présentant des cellules et utilisations associées
US9555105B2 (en) 2004-05-27 2017-01-31 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses thereof
US7754482B2 (en) 2004-05-27 2010-07-13 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses therefor
EP2295588A1 (fr) 2004-05-27 2011-03-16 The Trustees Of The University Of Pennsylvania Nouvel antigène artificiel présentant des cellules et utilisations associées
US8722400B2 (en) 2004-05-27 2014-05-13 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses therefor
US7608433B2 (en) 2005-02-09 2009-10-27 Idexx Laboratories Method of detection of classical swine fever
WO2007059513A2 (fr) 2005-11-15 2007-05-24 The Children's Hospital Of Philadelphia Compositions et procedes de modulation d’hemostase
EP2423224A1 (fr) 2005-11-15 2012-02-29 The Children's Hospital of Philadelphia Compositions et procédés de modulation de l'hémostase
EP2431389A1 (fr) 2005-11-15 2012-03-21 The Children's Hospital of Philadelphia Compositions et procédés de modulation de l'hémostase
EP2431390A1 (fr) 2005-11-15 2012-03-21 The Children's Hospital of Philadelphia Compositions et procédés de modulation de l'hémostase
WO2007068803A1 (fr) 2005-12-14 2007-06-21 Licentia Ltd Nouveau facteur neurotrophique et utilisations de celui-ci
EP2384764A2 (fr) 2006-02-23 2011-11-09 The Children's Hospital of Philadelphia Compositions et méthodes pour moduler une hémostase au moyen de différentes formes de facteur V activé
EP2479189A1 (fr) 2006-12-12 2012-07-25 Biorexis Pharmaceutical Corporation Bibliothèques de protéines hybrides de transferrine
EP2551281A1 (fr) 2007-10-25 2013-01-30 Nevalaita, Lina Variants d'épissage de GDNF et leurs utilisations
WO2009133247A1 (fr) 2008-04-30 2009-11-05 Licentia Oy Facteur neurotrophique manf et ses utilisations
WO2010060035A1 (fr) 2008-11-21 2010-05-27 The Children's Hospital Of Philadelphia Facteur v de serpent et procédés d'utilisation en tant que procoagulant
WO2012074868A2 (fr) 2010-12-03 2012-06-07 Ms Technologies, Llc Expression optimisée de molécules d'acide nucléique codant pour la résistance au glyphosate dans cellules végétales
WO2013049804A1 (fr) 2011-09-30 2013-04-04 The Children's Hospital Of Philadelphia Compositions et procédés pour moduler l'homéostasie
US9783817B2 (en) 2013-03-04 2017-10-10 Arkansas State University Methods of expressing and detecting activity of expansin in plant cells
WO2014191630A2 (fr) 2013-05-28 2014-12-04 Helsingin Yliopisto Modèle animal non humain codant pour un gène manf non fonctionnel
WO2018226887A1 (fr) 2017-06-07 2018-12-13 Spark Therapeutics, Inc. Agents d'activation destinés à la transfection de cellules et/ou la production de vecteur raav améliorées
WO2019152957A1 (fr) 2018-02-02 2019-08-08 Arizona Board Of Regents On Behalf Of Arizona State University Lymphocytes t de type adn-récepteur antigénique chimérique pour immunothérapie
WO2022079083A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acide nucléique pour transcription de va-arn
WO2022079082A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acides nucléiques améliorées pour activation de gènes simultanée
WO2023198685A1 (fr) 2022-04-13 2023-10-19 F. Hoffmann-La Roche Ag Procédé de détermination de génomes d'aav
WO2023227438A1 (fr) 2022-05-23 2023-11-30 F. Hoffmann-La Roche Ag Procédé raman de différenciation d'un sérotype de particules aav et d'un état de chargement de particules aav
WO2023232922A1 (fr) 2022-06-03 2023-12-07 F. Hoffmann-La Roche Ag Procédé de production de particules d'aav recombinées
WO2024013239A1 (fr) 2022-07-14 2024-01-18 F. Hoffmann-La Roche Ag Procédé de production de particules de virus adéno-associé recombinant
WO2024056561A1 (fr) 2022-09-12 2024-03-21 F. Hoffmann-La Roche Ag Procédé de séparation de particules de vaa pleines et vides
WO2024165456A1 (fr) 2023-02-07 2024-08-15 F. Hoffmann-La Roche Ag Procédé de détection d'anticorps anti-particules d'aav
WO2024194280A1 (fr) 2023-03-21 2024-09-26 F. Hoffmann-La Roche Ag Méthode destinée à produire des préparations de particules aav recombinantes

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